The incidence of extensively drug resistant (XDR) Mycobacterium tuberculosis (TB) is increasing at an alarming rate. Early and rapid detection of XDR TB could be crucial to disease management by alerting physicians to use salvage therapy and to institute special infection control procedures. This proposal seeks to leverage several successful programs in molecular diagnostics, sample processing, TB genetics and TB clinical trials at The New Jersey Medical School-UMDNJ, the NIH, and the biotechnology company Cepheid and the International Tuberculosis Research Center/National Masan Tuberculosis Hospital (NMTH) in Masan, Korea. Our collaborative goal is to transform detection of XDR TB from a months-long task into a 90- minute assay that can be performed directly on clinical sputum samples. We will develop a highly multiplexed XDR assay that uses a novel type of "sloppy molecular beacon" to perform multiplex melting temperature analysis and mutation detection. Reasons for discordances between conventional drug susceptibility testing and molecular assays will be investigated and the results used to make further assay improvements. An optimized assay incorporating all important XDR mutations will be tested, reduced to a format compatible with a simple sample-processing cartridge and then piloted on clinical samples in Masan. The specific aims of this proposal are to: 1) Develop a multiplexed molecular beacon-PCR assay to rapidly identify the common causes of TB resistance to FQs and injectable antibiotics. 2) Test the new assay on a library of TB samples, identify the causes of discordant results, and improve the assay coverage using same color multiplexing. 3) Reformat the XDR assay so that it can be performed directly on clinical sputum samples and detect XDR within two hours. Our overall goal is to transform XDR detection and management. PUBLIC HEALTH RELEVANCE: This project will also lead to a very rapid test to detect highly drug resistant tuberculosis so that it can be treated more rapidly and so that patients with this disease can be quickly isolated before they infect others.